The LNC-NINDS Protein/Peptide Sequencing Facility has continued to provide NINDS investigators with theoretical and technical expertise for the separation, purification, and amino acid sequencing of peptides and proteins. During the past year, 9 service mode amino acid sequencing projects have been completed, on peptides, fusion or endogenous proteins, or enzymatic or chemical fragments of proteins. Since its opening in 1922, 48 cooperative projects have been undertaken. Of these projects, which represent the main activity of the Facility, 20 were completed and 28 continued during the year. Highlights of three cooperative projects are described: (1) A 67 kDa cdc2-like kinase activator from rat spinal cord was isolated and purified. The protein was subjected to enzymatic degradation and the resulting peptide fragments purified by narrowbore RP-HPLC and sequenced. Sequences representing ca. 17% of the molecule were used to design the cDNA probes. Using oligonucleotide primers deduced from these amino acid sequences, full-length cDNAs have been obtained from rat brain libraries in conjunction with the techniques of RT-PCR and cDNA library screening. (2) Several proteins that may be involved in hibernation in the thirteen-lined ground squirrel (TLS) have been identified. A potential hibernation protein was submitted to the Facility as a 2D-PAGE PVDF blot and subjected to in situ proteolytic digestion. The protein was identified on the basis of a internal sequence as phosphoglycerase mutase. Plasma factors related to hibernation were purified by a combination of HP-SEC and RP-HPLC. Two proteins (30-70 kDa) elevated in the nonhibernating TLS plasma and essentially absent in the hibernating plasma, were identified by combination of N-terminal and internal amino acid sequencing as the previously reported HP-55 alpha-1 - antitrypsin and a previously unreported alpha-1-antitrypsin. The N- terminus of the unreported alpha-1-antitrypsin appears to significantly differ from others in the Swiss protein database, but is clearly placed in this family in the basis of its internal amino acid sequences. Two additional proteins were identified from lower molecular weight fractions. The first, is the known protein, apolipoprotein, and the second (elevated in the hibernating plasma), is a previously unreported protein, which upon proteolytic digestion yielded sequences, unmatched in the Swiss protein database. (3) In analogy to soluble CaM kinase II, postsynaptic density-associated CaM kinase II undergoes autophosphorylation in the presence of calcium/calmodulin. Thr (253) was identified as the major autophosphorylation site by sequencing of the radioactive peptides resulting from proteolytic cleavage of radiolabeled postsynaptic density-associated CaM kinase II. Significantly, phosphorylation at this site under similar conditions has not been reported in the soluble kinase.